Determination of free thyroid hormones by competitive immunoassay

ABSTRACT

A method is revealed for the quantitative determination of the proportion of the free form of a thyroid hormone ligand in a sample of a biological fluid in which the ligand is present partly in the free form and also partly in a form in which it is bound to physiological binding proteins. In the first step, a ligand derivative of the thyroid hormone with an immunoglobulin, a labelled specific binder, and a test tube whose walls are coated with an excess of a protein material are provided. In the second step, a sample of the biological fluid containing an unknown amount of the free thyroid hormone ligand, a solution containing a known amount of the ligand derivative, and a solution containing a known, less than stoichiometric amount of the labelled specific binder are added to the test tube to form a liquid reaction mixture. In the third step, the reaction mixture is incubated to allow the free thyroid hormone ligand and the ligand derivative in the liquid reaction mixture to compete for the binding sites of the labelled specific binder, and to allow, in a concomitant slower reaction, the binding of the immunoglobulin part of the ligand derivative to the excess of the protein material on the walls of the test tube. In the fourth step, the remaining liquid reaction mixture is removed from the test tube. In the final steps, the amount of labelled specific binder bound to the walls of the test tube is determined and the amount of the free thyroid hormone ligand calculated.

The present invention relates to a method for the determination of theamount of a ligand in a sample of a biological fluid, in which thebiological fluid containing the ligand to be determined is incubated inthe presence of a dissolved ligand derivative, which can be convertedinto an insoluble form, in the liquid phase with less than thestoichiometric amount of a labelled specific binder which binds both theligand to be determined and the ligand derivative so that the ligand tobe determined and the ligand derivative compete for the labelledspecific binder, and in which the ligand derivative is converted into aninsoluble form and, after separation of the insoluble components of theassay system from those components of the assay system remaining in theliquid phase, the amount of the ligand to be determined in thebiological fluid is calculated from the amount of the labelled specificbinder bound to the ligand derivative, and a kit for carrying out such amethod.

The present invention relates in particular to a method of the statedtype for the determination of the free portion of a monovalent ligand,i.e. of a ligand which represents a monovalent antigen or a hapten,which is to say that it has only one binding site for the immunologicalbinding by a specific binding partner, namely a thyroid hormone, whichis present in biological fluids partly in a form bound to itsphysiological binding proteins and partly in unbound, i.e. free, form,and the object of the process is the quantitative determination of thisfree proportion.

The method according to the invention is one of the immunological assaymethods in which labelled antibodies (labelled specific binders) areemployed and which are also referred to as immunometric assay methods.It belongs to the version of such methods which is referred to as acompetitive method. In contrast to typical sandwich methods, in whichthe total amount of the ligand to be determined is bound in a sandwich,a proportion of the labelled binding partner is bound to the ligand tobe determined and a further proportion to a competing derivative of thisligand. Further statements on various types of immunometric assaymethods are to be found in the introduction of EP-B-89806 in which,exactly as in EP-B-103605, the immunometric version of a method for thedetermination of free proportions of ligands is described, which methodis described as the so-called analogue tracer assay method inEP-B-26103.

In immunometric methods of the type according to the invention for thedetermination of the free proportions of ligands, in particularmonovalent ligands such as the thyroid hormones, the ligand to bedetermined and the ligand derivative taken in a known amount are allowedto compete for a minor amount of the labelled antibodies according tothe above description of the fundamental method on which the presentinvention is based. The labelled antibody is bound essentiallycompletely to the free ligand and the ligand derivative, and itsdistribution over the two binding partners reflects the concentration tobe determined of the ligand in the sample. In order to obtaininformation about the concentration to be determined for the ligand andthe distribution of the labelled antibody over the ligand to be measuredand the ligand derivative, the reaction product of ligand derivative andlabelled antibody is preferably immobilised for separation from theremaining assay solution, which, according to EP-P-103605, is effectedby converting the ligand derivative into an insoluble form before orafter incubation with the labelled antibody. In practice, the proceduregenerally adopted to date is to initially take the ligand derivativefrom the outset in a form immobilised by binding to the solid substrate.

Embodiments of a method of this type are described in EP-A-303284 andEP-A-324540.

In a version of the method of the stated type, which version isdescribed in EP-A-149631, an exogenous binder for the ligand derivativeis used in addition to the conventional components of the assay method.This exogenous binder for the ligand derivative may be an additionalantibody specific to the latter. Its function should be to form a sortof buffer for the ligand derivative. Since, in the method discussed inEP-A-149631, the ligand derivatives are chemically modified ligands andhave hapten character and are therefore monovalent, in the methodaccording to EP-A-149631 the exogenous binder does not bind, withimmobilisation, the complex formed in the determination and consistingof ligand derivative and labelled antibody.

In another method for the determination of free substances according toEP-A-106615, a specific binder is used which simultaneously binds to theligand to be determined and to an antibody for the specific binder, andthe binding of the ligand and the binding of the additional antibodyshould influence one another so that there is competition between thetwo and it is possible to derive the concentration of the free ligand tobe determined from the extent of binding of the antibody.

If, in methods according to EP-B-89806 or EP-B-103605 or EP-A-303284 orEP-A-324540, immobilised ligand derivatives which compete with the freeligand to be determined for the labelled antibody are used, problems("matrix effects") are frequently encountered in practice and are due tothe fact that the reaction between the immobilised ligand derivative andthe labelled antibody are reactions at the surface of the solid phasewhere diffusion processes play an important role and which areconsiderably determined by certain random properties of the solid phase.In practice, these matrix effects mean that the individual productionbatches of solid substrates with the immobilised ligand derivatives mustall be carefully tested for their properties, with the frequentoccurrence of high percentages of useless substrates which have to beseparated as waste and which considerably increase the costs of theproduction process and therefore also of the final assay method.

EP-A-105 714 furthermore discloses that, in typical immunometricsandwich methods in which a polyvalent ligand, such as, for example,TSH, is completely bound by reaction with two different antibodies, therequired incubation times can be shortened by carrying out the actualsandwich formation as a liquid-phase reaction and then extracting thesandwich formed from the reaction system by adding a solid phase havinga binding partner for one of the sandwich antibodies. This method issuitable only for the determination of the total concentration of aligand which has at least two binding sites for specific bindingpartners.

It is an object of the present invention to provide a rapid and reliablemethod for the determination of the amount of a free ligand in a sampleof a biological fluid according to the precharacterising clause ofPatent Claim 1, which can be carried out rapidly, reliably and with highaccuracy and in which the matrix problems described above, whichadversely affect the cost-efficiency of the conventional correspondingmethods, are avoided.

This object is achieved with a method according to the precharacterisingclause of Patent Claim 1 by the measures stated in the characterisingclause of this Claim.

Advantageous and preferred embodiments of a process of this type and akit which serves for realising the method are described in thesubclaims.

The procedure adopted in the method according to the invention is suchthat the competing reaction of the free ligand to be determined and ofthe ligand derivative with the labelled antibody takes place in theliquid phase in the presence of a protein material which is bound to asolid phase and which specifically binds the substrate protein part of aligand derivative used as a conjugate in a reaction which is slower thanthe actual immune reaction and leads to a non-covalent protein bond, andthus extracts the immune complex comprising ligand derivative andlabelled antibody from the liquid reaction mixture.

In the method according to the invention, at the end of the reaction thelabelled antibody bound to the ligand derivative is bound as part of asandwich structure via the ligand derivative, its substrate protein partand the immobilised protein material to the solid substrate. In contrastto conventional sandwich methods, what is therefore bound is the ligandderivative used in a predetermined amount and not the ligand to bedetermined. In the case of monovalent ligands, such as monovalentantigens or haptens, such as, for example, the thyroid hormones, it isusually not possible to produce a sandwich structure. This is, however,permitted in accordance with a method as disclosed in EP-A-161 107 bythe use of a soluble ligand derivative in the form of a conjugate of aligand with a substrate protein. The presence of the free ligand to bedetermined is evident as a disturbance of the synthesis of the statedsandwich structure, in that those parts of labelled antibodies which arebound by the free ligand are not immobilised on the solid phase butremain in solution and therefore provide information about the amount ofthe ligand to be determined.

In the present application--and, for example, also in EP-A-303284 --aderivative of the ligand, in which the latter or a chemically modifiedform thereof is covalently bonded to a protein substrate molecule, isdefined as a conjugate of a ligand.

By using a ligand derivative which is a conjugate of the ligand with asterically bulky substrate protein, the ligand derivative issimultaneously a "differentially binding ligand derivative" in themeaning of the prior art described at the outset, which derivative hasconsiderably reduced binding capability compared with physiologicalbinding proteins and hence permits trouble-free determination of thefree proportion of a ligand to be determined.

The labelled antibody used in the method according to the invention ispreferably a monoclonal antibody which has such affinity properties withrespect to the free ligand to be determined and the ligand derivativethat it is bound to both to an extent which reflects the amount of thefree ligand to be determined, and different affinities may be capable ofcompensating for the effect of relatively large concentrationdifferences of the two binding partners. In the case of thedetermination of free thyroxine (FT4), for example, a monoclonalantibody having an association constant of the order of 10¹⁰ l/mol issuitable.

The ligand derivative is as a rule used in an amount which is of theorder of 0.5 to 20 times the molar amount, based on the amount of theligand to be determined which is to be expected in a sample from anormal patient. The labelled antibody is used in an amount which is lessthan the stoichiometric amount based on the sum of the amount of theligand to be determined which is to be expected in the sample and theknown added amount of the ligand derivative.

The method according to the invention is described in detail below,taking as an example a preferred embodiment in the form of a coated tubetest method for the determination of free thyroxine (FT4) in the serum.In this method, a constant, predetermined amount of a T4-IgG conjugate(L-IgG) and free T4 (L) from the sample compete in the solution for aconstant amount of labelled anti-T4 antibody (tracer; Ak*). Immobilisedon the tube walls is an anti-IgG antibody (anti-IgG) which binds the T₄-IgG conjugate and hence, as part of the immune complex comprisingligand derivative and tracer, also the tracer part which is not bound bythe fractions of free T₄ present in the sample and is therefore nolonger available for binding to the T₄ -IgG conjugate. The measurementis thus based on an FT₄ concentration-dependent disturbance of theimmunometric determination of a constant amount of a T₄ derivative bysandwich formation.

The labelled antibody can be labelled with any of the known labels orlabel systems which are listed in detail, for example, in the prior artpublications discussed at the outset. In particular, it can be labelledwith a known radioactive isotope, in particular an iodine isotope, andthe assay method is then an immunoradiometric assay method. The labelledcomponent can, however, also be another known label, for example anenzyme or a fluorescent or, preferably, a chemiluminescent label.

The method according to the invention is illustrated in detail below fora preferred embodiment with reference to the Figures.

FIG. 1 shows a schematic representation of the test principle;

FIG. 2 shows an elution profile of an HPLC gel chromatography of areaction mixture for preparation of the T₄ -IgG conjugate used as theligand derivative in a preferred embodiment of the method according tothe invention, the natural T₄ -IgG conjugate used in the Examplesdescribed below being eluted at 11 minutes;

FIG. 3 shows a typical standard curve for the method according to theinvention for the determination of FT₄ ; and

FIG. 4 shows a comparison of the results of the method according to theinvention with those of a commercially available assay method for FT₄(DYNOtest FT₄ from Henning Berlin).

In the method described as the preferred embodiment, according to thegenerally valid scheme shown in FIG. 1, the serum sample containing theligand to be determined, a T₄ rabbit IgG conjugate solution and asolution containing labelled anti-T₄ antibodies are pipetted immediatelyin succession into a test tube coated with goat anti-rabbit antigens.The labelled anti-T₄ antibody is preferably a monoclonal antibody andcross-reacts neither with the substrate protein part of the T₄ -IgGconjugate nor with the coating of the test tube. FT₄ from the samplethus competes with the T₄ rabbit IgG conjugate as the ligand derivativefor a labelled specific binder in the form of a labelled anti-T₄antibody, in a homogeneous liquid-phase reaction. At the same time, butmore slowly, T₄ rabbit IgG conjugate is bound to the immobilised goatanti-rabbit antibody and--inversely proportionally to the FT₄ content ofthe serum sample--conjugate-bound, labelled anti-T₄ antibody. Afterincubation for two hours, the test tube is washed and the labelledantibody remaining in the tube is measured on the basis of itslabelling.

Since the decisive competitive reaction in the liquid phase takes placesubstantially more rapidly than the liquid-solid phase reaction, thismethod according to the invention does not have the fundamental kineticproblems of a corresponding method in which a T₄ conjugate immobilisedat the outset is employed.

For the described version of the method according to the invention, inthe preparation of the T₄ rabbit IgG conjugate used as a ligandderivative, it is necessary to ensure the immunological recognition ofsaid conjugate both with respect to the T₄ part by the labelled antibodyand with respect to the IgG part for immobilisation by production of asandwich.

This requirement can be met, for example, if, as in the Example below,the T₄ rabbit IgG conjugate used as the ligand derivative is obtained bya gentle method in which the substrate protein is not significantlydenatured or which permits isolation of conjugates with a natural,nondenatured protein moiety.

EXAMPLE

a) Preparation of a T₄ rabbit IgG conjugate

34μg of T₄ -NHS active ester (Henning Berlin) in 50 μl of acetonitrileare incubated with 1 mg of rabbit IgG (SIGMA) in 0.5 ml of phosphatebuffer, pH 8.0, for 90 minutes at room temperature. Natural rabbit IgGreacted with T₄ -NHS active ester is isolated by HPLC gel chromatographyand thus separated from T₄ -IgG aggregates, unreacted T₄ -NHS activeester and other reaction products. The example of an elution profile isshown in FIG. 2. The T₄ -IgG loading is determined by means of amultichannel UV absorption detector continuously measuring the columnflow, through measurement of the absorbance at 325 and 280 nm during thechromatography.

In the determination, the amount of T₄ rabbit IgG conjugate perdetermination which is used in the test is the maximum of 20 times the(molar) amount of FT4 in a sample in a normal patient (about 20 pmol/l).

b) Preparation of a labelled anti-T₄ antibody

As a labelled specific binder, a monoclonal anti-T₄ antibody obtained ina manner known per se is labelled by a known reaction with an acridiniumester as a chemiluminescent label. The labelled antibody is used in thetest method in a substoichiometric amount relative to the T₄ rabbit IgGconjugate and relative to FT₄. Luminescent labelling of the antibody iscarried out as follows:

10 μg of acridinium ester in 10 μl of acetonitrile (Hoechst Behring) areincubated with 100 μg of monoclonal anti-T₄ antibody (Henning Berlin) in90 μl of phosphate buffer, pH 8.0, for 20 minutes. The labelled antibodyis then purified by hydroxyapatite chromatography. About 110,000 RLU(measured with an Autoclinilumat from Berthold) of the labelled antibodyare used per determination.

c) Preparation of the test tubes coated with the protein material

The tubes used in the test are prepared by coating suitable polystyrenetubes with 2.5 μg of goat anti-rabbit IgG (SCANTIBODIES)/0.5 ml of 0.1MNaHCO₃, pH 8.0, for 24 hours at room temperature. This is followed bydecanting, washing with water and subsequent coating for two hours with3% solution of a sorbitol syrup in which crystallisation has beensuppressed (trade mark Karion), 0.5% of BSA and 0.005% of NaN₃.Decanting is carried out again and the tubes are freeze-dried forstorage for subsequent use.

d) Assay procedure

The test is carried out as follows, using the components prepared aboveunder a) to c):

The following are pipetted immediately in succession per tube: 50 μl ofserum sample or standard, 200 μl of T₄ -IgG conjugate solution, 200 μlof solution containing labelled antibody. The two last-mentionedsolutions contain 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% gelatine and0.05% NaN₃. The tubes are incubated for 2 hours while shaking at 170rpm. Finally, the tubes are each washed with 4×1 ml of wash solution andare measured in a luminometer. The following results illustrated byFIGS. 3 and 4 are obtained:

FIG. 3 shows the average standard curve of the method described, whichcurve results from the determination of the inter-assay variation of thetest.

FIG. 4 shows the correlation, obtained with 189 patient sera, betweenthe test according to the invention and a known commercial test(DYNOtest FT₄ from Henning Berlin). The evaluation using a computerprogram gave a coefficient of 0.96. The line of best fit was calculatedas follows:

(FT₄ test according to the invention; pmol/l)=0.8×(DYNOtest FT4;pmol/1)+1.1 pmol/l.

e) Further testing of the effect of certain assay parameters on theassay method

e1) Effect of the variation of the amount of T₄ rabbit IgG conjugateused at various T₄ -IgG loading levels:

Four different T₄ -IgG conjugates were synthesised and purified, and theT₄ loading was determined by measuring the absorbances at 325 nm and 280nm. Table 1 below shows that only very small amounts of conjugate arerequired in order to achieve binding of the tracer in the FT₄ testaccording to the invention.

                  TABLE 1                                                         ______________________________________                                                 T.sub.4 -IgG loading                                                                        Amount of con-                                                  level         jugate per                                             Conjugate #                                                                            325 nm/280    determination                                                                             % B.sub.o T                                ______________________________________                                        1        3.9 10E-2     20 ng       49                                         1        3.9 10E-2     5 ng        19                                         1        3.9 10E-2     1 ng         5                                         2        2.3 10E-2     20 ng       32                                         2        2.3 10E-2     5 ng        11                                         2        2.3 10E-2     1 ng         3                                         3        1.5 10E-2     20 ng       21                                         3        1.5 10E-2     5 ng         7                                         3        1.5 10E-2     1 ng         2                                         4        1.1 10E-2     20 ng       15                                         4        1.1 10E-2     5 ng         5                                         4        1.1 10E-2     1 ng         2                                         ______________________________________                                    

e2) Testing of the stability of the test tubes used in the FT₄ testaccording to the invention and coated with anti-rabbit IgG:

The change in the quality of the tubes due to storage was tested byincubation with ¹²⁵ I-labelled rabbit IgG and the same amount of ¹²⁵I-labelled rabbit IgG which had been diluted with an excess ofunlabelled rabbit IgG. Table 2 below shows the binding found in thepresence of the unlabelled rabbit IgG, based on the binding of theundiluted ¹²⁵ I-labelled rabbit IgG, and the coefficient of variance of50 determinations of this binding.

                  TABLE 2                                                         ______________________________________                                                  % binding + IQG addition,                                           Storage after                                                                           based on binding without                                                                        % CV                                              production                                                                              IgG addition      (50 times)                                        ______________________________________                                        1 day     45.4              2.4                                               5 weeks, 4° C.                                                                   45.3              2.1                                               5 weeks, 37° C.                                                                  43.6              1.8                                               ______________________________________                                    

The results show that the tubes have very high stability--even atelevated storage temperatures. These stabilities are considerably betterthan those of conventional test tubes on whose walls an immobilised T₄derivative in the form of a T₄ -IgG conjugate is present. In these knowntubes, considerable changes occur after storage for only one week at 37°C., in comparison with the data for the freshly produced tubes. Incontrast, the tubes to be used in the method according to the inventionare stable over several weeks at 37° C.

We claim:
 1. A method for the quantitative determination of the amountof free form of a thyroid hormone ligand in a sample of a biologicalfluid in which the ligand is present partly in the free form and alsopartly in a form in which it is bound to physiological binding proteins,comprising the steps of:a) providing(i) a ligand derivative in the formof a soluble conjugate of the thyroid hormone with an immunoglobulin,the ligand derivative having essentially no binding capability withrespect to the physiological binding proteins, (ii) a labelled specificbinder which in an immunological binding reaction specifically bindsboth the free thyroid hormone ligand and the ligand derivative, and(iii) test tubes whose walls are coated with an excess of a proteinmaterial which selectively binds the immunoglobulin part of the ligandderivative without affecting the binding between the thyroid hormonepart of the ligand derivative and the labelled specific binder, theprotein material being selected from anti-isotypic immunoglobulins andanti-isotypic poly- or monoclonal antibodies against the immunoglobulinpart of the ligand derivative, b) adding to the test tubesi) a sample ofthe biological fluid containing an unknown amount of the free thyroidhormone ligand, or a standard solution of the thyroid hormone ligand,ii) a solution containing a known amount of the ligand derivative, andiii) a solution containing a known, less than stoichiometric amount ofthe labelled specific binder, to form a liquid reaction mixture; c)incubating the reaction mixture to allow the free thyroid hormone ligandand the ligand derivative in the liquid reaction mixture to compete forbinding sites of the labelled specific binder, and to allow, in aconcomitant slower reaction, binding of the immunoglobulin part of theligand derivative to the excess of the protein material on the walls ofthe test tubes; d) removing remaining liquid reaction mixture from thetest tubes; e) determining the amount of labelled specific binder boundto the walls of the test tubes; and f) calculating the amount of thefree thyroid hormone ligand.
 2. The method of claim 1, wherein the freethyroid hormone ligand to be determined is free thyroxine (FT₄), theligand derivative is a thyroxine-IgG conjugate and the protein materialbound to the walls of the test tubes is an anti-isotypic IgG.
 3. Themethod of claim 2, wherein the ligand derivative is a thyroxine-rabbitIgG conjugate and the anti-isotypic IgG is a goat anti-rabbit IgG or agoat anti-rabbit antibody.
 4. The method of claim 1, wherein the wallsof the test tubes are coated with an amount of the protein materialwhich is at least sufficient for completely binding the total amount ofthe ligand derivative used,wherein an amount of the ligand derivativeused is between 0.5 to 20 times the molar amount of free thyroid hormoneligand expected in a normal patient, and wherein the labelled specificbinder is used in a sub-stoichiometric amount such that only a portionof the free ligand present in the sample and of the ligand derivativereact with the labelled specific binder.
 5. The method of claim 2,wherein said labelled specific binder is a labelled monoclonal antibodyto T₄ which does not react neither with the immunoglobulin part of theligand derivative nor the protein material.
 6. The method of claim 1,wherein the immunoglobulin part of the ligand derivative is a nativeimmunoglobulin.
 7. The method of claim 1, which is carried out in thefollowing sequence of incubation steps:a) adding to the test tubes thesample of the biological fluid, or the standard solution of the thyroidhormone ligand; b) adding a solution which contains the known amount ofthe ligand derivative; and c) adding the solution containing the known,less than stoichiometric amount of the labelled specific binder; and, d)after an adequate incubation period and removal of the liquid reactionmixture from the test tubes, washing the test tubes and determining theamount of free thyroid hormone ligand in the sample on the basis ofproportion of labelled specific binder bound to the walls of the testtubes, referring to results obtained by same method for standardsamples.
 8. A kit for carrying out a method for quantitativedetermination of the amount of free form of a thyroid hormone ligand ina sample of a biological fluid, in which the ligand is present partly inthe free form and also partly in a form in which it is bound tophysiological binding proteins, which kit comprises, as separatereagents:i) a known amount of a ligand derivative in form of a solubleimmunoglobulin conjugate of the thyroid hormone ligand, ii) a knownamount of a labelled anti-ligand antibody, iii) test tubes whose wallsare coated with an excess of an anti-isotypic immunoglobulin against theimmunoglobulin part of the ligand derivative; and, optionally, iv)standard solutions containing known amounts of the thyroid hormoneligand to be determined, and v) buffer and dilution solutions.
 9. Thekit of claim 8, wherein the ligand derivative, the labelled anti-ligandantibody, the coating of the test tubes, and, optionally, the standardsamples are present in freeze-dried form.